1. Field of the Invention
This invention relates to isolated proteins or peptides useful for inhibition of matrix metalloproteinases. Specifically, this invention relates to a novel protein, isolated from conditioned media of cultured human tumor cells, which binds with high affinity to matrix metalloproteinase enzymes and analogs thereof. The natural protein is defined by a novel amino acid sequence including specific positions of cysteine residues. This invention further relates to a novel means of purifying matrix metalloproteinase inhibitors using metalloproteinase affinity chromatography.
2. Background
The collagenase family of enzymes are a group of neutral metalloproteinases, also known as matrix matalloproteinases, which are secreted in the zymogen form and degrade both the collagenous and noncollagenous components of the extracellular matrix. All require a metal ion (calcium and/or zinc) for hydrolytic activity, and all are secreted in the latent pre-enzyme form. Members of this collagenase gene family include: the interstitial collagenases, which degrade collagen types I, II and III and have been characterized with respect to substrate specificity and requirements for activation (Stricklin, G. P., Jeffrey, J. J., Rosewit, W. T., and Eisen, A. Z., 1983, Biochemistry 22, 61-68; Goldberg, G. I., Wilhelm, S., Kronberger, A., Bauer, E. A., Grant, G. A., and Eisen, A. Z., 1986, J. Biol. Chem. 261, 6600-6605; Hasty, K. A., Jeffrey, J. J., Hibbs, M. S., and Welgus, H. G., 1987, J. Biol. Chem. 262, 10048-1052; Fields, G. B., Van Wart, H. E., and Birkedal-Hansen, H., 1987, J. Biol. Chem. 262, 6221-6226; Grant, G. A., Eisen, A. Z., Marmer, B. L. Rosweit, W. T., and Goldberg, G. I., 1987, J. Biol. Chem. 262, 5886-5889); stromelysin, which degrades proteoglycans, glycoproteins, and the non-helical portions of collagenous molecules (Wilhelm, S. M., Collierm, I. E., Kronberger, A., Eisen, A. Z., Marmer, B. L., Grant, G. A., Bauer, E., and Goldberg, G. I., 1987, Proc. Natl. Acad. Sci. U.S.A. 84, 6725-6729; Whitman, S. E., Murphy, G., Angel, P., Rahmsforf, H. J., SMith, B. J., Lyons, A., Harris, T. J. T., Reynolds. J. J., Herrlich, P. and Docherty, A. J. P., 1986, Biochem. J. 240, 913-916); and type IV collagenase, which degrades pepsin-resistant triple-helical type IV collagen and interstitial collagens (gelatin). Type IV collagenase has been identified in human tumor cells (Liotta, L. A., Kleinerman, J, Catanzaro, P., and Rynbrandt, D., 1977, J. Natl. Cancer Inst. 58, 1427-1439; Turpeenniemi-Hujanen T., and Tryggvason, K., 1982, Int. J. Cancer 30p, 669-673; Liotta, L. A., Abe, S., Gehron-Robey, P., and Martin, G. R., 1979, Proc. Natl. Acad. Sci. U.S.A. 76 268-2272; Liotta, L. A., Tryggvasson, K., Garbisa, S., Hart, I., Foltz, C. M., and Shafie, S., 1980, Nature (London) 284, 67-68; Collier, I. E., Wilhelm, S. M., Eisen, A. Z., Mariner, B. L., Grant, G. A., Seltzer, J. L., Kronberger, A., He., C., Bauer, E. A., and Goldberg, G. I., 1988, J. Biol. Chem. 263, 6579-6587) , endothelial cells (Kalebic, T., Barbisa, S., Glaser, B., and Liotta, L. A., 1983, Science 221, 281-283), bone (Murphy, G., McAlpine, C. G., Poll, C. T., and Reynolds, J. J., 1985, Biochem. Biophys. Acta 831, 49-58), fibroblasts (Collier, I. E., Wilhelm, S. M., Eisen, A. Z., Marmer, B. L., Grant, G. A., Seltzer, J. L., Kronberger, A., He., C., Bauer, E. A., and Goldberg, G. I., 1988, J. Biol. Chem. 263, 6579-6587), polymorphonuclear leukocytes (Uitto, V. J., Schwartz D., and Veis, A., 1980, Eur. J. Biochem. 105, 409-417) and macrophages (Garbidsa, S., Ballin, M., Daga-Giordini, D., Fastelli, G., Naturale, M., Negro, A., Semenzato, G., and Liotta, L. A., 1986, J. Biol. Chem. 261, 2369-2375). This enzyme is a neutral metalloproteinase of 68 to 72 kilodaltons which is secreted in zymogen form (Liotta, L. A., Abe, S., Gehron-Robey, P., and Martin, G. R., 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 2268-2272; Liotta, L. A., Tryggvassin, K., Garbisa, S., Gehron-Robey, P., and Abe, S., 1981, Biochemistry 20, 100-104; Salo, T., Liotta, L. A., and Tryggvsasson, K., 1983, J. Biol. Chem. 258, 3058-3063). In addition, several other members of this collagenase gene family have been described recently, including a second type of stromelysin (stromelysin-2), a 92 kilodalton form of type IV collagenase, and Putative Uterine Metalloproteinase (PUMP)-1, a low molecular weight uterine collagenase (Wilhelm, S. M., Collier, I. E., Marmer, B. L., Eisen, A. Z., Grant, G. A., and Goldberg, G. I., 1989, J. Biol. Chem. 264, 17213-17221; Woessner, J. F. and Talpin, C. J., 1988, J. Biol. Chem. 263, 16918-16925).
The matrix metalloproteinases are thought to play an important role in disease processes characterized by the inappropriate destruction of the extracellular matrix. The diseases include inflammatory processes such as rheumatoid arthritis and other autoimmune disorders, tumor cell invasion and metastasis formation, local sequelae of myocardial anoxia, and corneal ulceration (Okada et al., 1986, J. Biol. Chem. 261, 14245-14255; Harris et al., 1984, Collagen Relat. 4, 493-512; Werb et al., 1977, New Engl. J. Med. 296, 1017-1023; Liotta et al., 1980, Nature (London) 284, 67-68; Kalebic et al., 1983, Science 221, 281-283). Many tissues contain natural inhibitors of the matrix metalloproteinases. In some cases, this inhibitory activity is derived from the antiproteases in plasma, particularly .alpha..sub.2 -macroglobulin and .beta..sub.1 -anticollagenase. .alpha..sub.2 - Macroglobulin is a high molecular weight (725,000 Da) inhibitor present in serum. It is thought to account for 95% of the collagenolytic inhibitory activity present in serum. Because of its large size, it is normally unable to pass the vascular permeability barrier. Under conditions of extreme inflammation in which there is increased capillary permeability, .alpha..sub.2 -macroglobulin may enter the tissue compartments and play a role in the regulation of matrix metalloproteinases. The mechanism of inhibition of the matrix metalloproteinases by .alpha..sub.2 -macroglobulin has not been directly studied. However, it is thought to be similar to the mechanism whereby .alpha..sub.2 -macroglobulin causes inhibition of other proteases. Hence, the mechanism is not believed to be unique for the matrix metalloproteinases.
.beta..sub.1 -Anticollagenase is approximately 40,000 daltons in size. It accounts for approximately 5% of the metalloproteinase inhibiting activity of serum. This inhibitor is thought to pass the vascular permeability barrier and be widely distributed in the tissue compartments. .beta..sub.1 -Anticollagenase may be related to another group of natural inhibitors of the metalloproteinases referred to as TIMPs, tissue inhibitors of metalloproteinases. The down-regulation of metalloproteinase collagenolysis and proteolysis may occur through TIMPs.
The prototype TIMP, TIMP-1 , is a glycoprotein with an apparent molecular size of 28.5 kDa which forms a complex of 1:1 stoichiometry with activated interstitial collagenase, stromelysin, and the 92 kDa type IV collagenase (Welgus and Stricklin, 1983, J. Biol. Chem. 253, 12259-12264: Welgus et al., 1985a, Collagen Rel. Res. 5, 167-179; Wilhelm et al., 1989, J. Biol. Chem. 264, 17213-17221; European patent 189,784). The gene coding for TIMP-1 has been cloned, sequenced and mapped to the X-chromosome (Carmichael et al., 1986 , Proc. Natl. Sci. USA 83, 2407-2411; Docherty et al., 1985, Nature (London) 318, 66-69; Mullins et al., 1988, Genomics 3, 187-194; Mahtani et al., 1988, Genomics 2, 294-301). The secreted protein has 184 amino acids and six intramolecular disulfide bonds. Reduction and alkylation of TIMP-1 abolishes all inhibitory activity. The same cells which produce interstitial collagenase are capable of synthesizing and secreting TIMP-1 (Welgus et al., 1985b, J. Clin. Invest. 76, 219-224; Herron et al., 1986, J. Biol. Chem. 261, 2814-2818). Thus, the net collagenolytic activity for these cell types is the result of the balance between activated enzyme levels and TIMP-1 levels. Studies have shown an inverse correlation between TIMP-1 levels and the invasive potential of murine and human tumor cells. Down-modulation of TIMP-1 mRNA levels by use of TIMP-1 antisense RNA resulted in conversion of previously nontumorigenic, noninvasive Swiss 3T3 cells to tumorigenic cells with invasive properties in vitro and metastatic potential in vivo (Khokha et al., 1989, Science 243, 947-950).
Another class of biologically active collagenase inhibitors is composed of low molecular weight (&gt;10,000 daltons) cationic proteins isolated from cartilage, aorta and teeth, but which are poorly characterized.
Recently several new members of the matrix metalloproteinase family have been identified, with various substrate specificities. These include stromelysin (homologue of rat transin), type IV collagenase (70 kDa gelatinase) and a 92 kDa gelatinase. While also identified in normal cell types, the over-expression of these enzymes has been linked to malignant conversion and the metastatic phenotype in a number of systems. Thus, there is a need to understand the molecular basis of the regulation of these metalloproteinases and to find inhibitors which can be exploited for diagnostic and therapeutic purposes.